Plastids are self-replicating organelles containing their own DNA in a single circular chromosome, called their genome. Plastids are found in all plant cells. They are inherited maternally in most plants just like mitochondria in animals and plants. This is also called cytoplasmic inheritance since these organelles are present in the cytosol of the ova.
Plant plastids (e.g. chloroplasts, amyloplasts, elaioplasts, etioplasts, chromoplasts, leucoplasts and proplastids) are the organelles in which major biochemical processes (i.e. photosynthesis) take place. In general, plant cells contain between 100-10,000 copies of the small 120-160 kb circular plastid genome. Since each molecule has one inverted repeat it is theoretically possible to obtain plant cells with 20,000 copies of (a) gene(s) of interest, after plastid transformation.
The genetic transformation of the plastid genome (plastome) has major advantages over nuclear transformation. Firstly, because in most plant species, plastids are maternally inherited, out-crossing of transgenes to weeds or other crops is minimized. Thus, this form of genetic engineering of plants lowers the risk of dissemination of the transgene in the environment through pollen dispersal. Furthermore, the plastid genome is highly polyploid, enabling the introduction of many copies per cell which can lead to high accumulation levels of the desired protein(s). The fact that plastids are able to form disulfide bonds and to fold proteins, makes this technique in theory ready for the production of biopharmaceuticals in plants.
The principle of plastid transformation is insertion of sequences through homologous recombination. Plastid transformation vectors use two targeting DNA segments that flank the gene or genes of interest. By means of homologous recombination these segments can insert the foreign gene or genes at a precise, predetermined position in the plastid genome. Position effects and gene silencing, major problems in nuclear transformation experiments, have not as yet been observed in plastid transformation events.
However, successful chloroplast transformation of crop plants is described thus far only for Solanaceous crops like potato, tomato, tobacco (U.S. Pat. No. 5,451,513; Svab et al. (1990), Proc. Natl. Acad. Sci. USA 87:8526-8530) and Brassicaceae, like Arabidopsis thaliana (U.S. Pat. No. 6,376,744). It is not obvious that the techniques used for these species can be readily used for other species such as Asteraceae, in particular lettuce.
It is therefore the object of the invention to provide an alternative plastid transformation method that is in particular useful for transforming Asteraceae plant species, such as lettuce (Lactuca sativa). Lettuce is an agronomical important crop and a useful transformation method therefore is thus highly desirable.